Recombinant C140 receptor, its agonists and antagonists, and nucleic acids encoding the receptor

ABSTRACT

Nucleic acid molecules encoding the C140 cell surface receptor have been cloned and sequenced. The availability of C140 receptor DNA permits the recombinant production of the C140 receptor which can be produced on the surface of a cell, including an oocyte. The nucleic acid molecules are useful in an assay for detecting a substance which affects C140 receptor activity, either receptor agonists or antagonists. Further, the elucidation of the structure of the C140 receptor permits the design of agonist and antagonist compounds which are useful in such assays. The availability of the C140 receptor also permits production of antibodies specifically immunoreactive with one or more antigenic epitopes of the C140 receptor.

TECHNICAL FIELD

[0001] The invention relates to a newly discovered receptor which is amember of the G-protein-coupled receptor superfamily. The receptor isexpressed in endothelial cells in blood vessels. Avoidance of effects onthis receptor is an essential element in limiting side effects of drugswhich are administered to stimulate other receptors in this family. Theinvention also relates to nucleic acid sequences encoding the receptorprotein or peptide.

BACKGROUND ART

[0002] Responses of animals to many therapeutic and prophylactic drugsare mediated through receptors which reside on cell surfaces. One classof such receptors comprises the G-protein-coupled receptors, whosephysiological effect is mediated by a three-subunit protein complex,called G-proteins, that binds to this type of receptor with thesubsequent release of a subunit, thus setting in motion additionalintracellular events. Receptors of this subclass include, among others,adrenergic receptors, neuropeptide receptors, the thrombin receptor andthe C140 receptor which is the subject of the herein invention. Thisclass of receptor is characterized by the presence of seventransmembrane regions which anchor the receptor within the cell surface.

[0003] It is the elusive goal of the designers of therapeutic substancesto effect a desired response in a subject in the absence of sideeffects. Accordingly, pharmaceuticals designed to target a specificreceptor, such as the thrombin receptor, should react with the thrombinreceptor specifically and have no effect on related receptors. The C140receptor of the present invention may be involved in controllingvascular pressure, and inadvertent stimulation or blocking of thisreceptor would have unpredictable and therefore undesirable results. Itis therefore useful to determine in advance whether therapeutic reagentsdesigned to target, for example, the thrombin receptor will or will nothave the undesired side effect of reactivity with the C140 receptor. Byproviding the recombinant materials for the production of the C140receptor in convenient assay systems, as well as agonist and antagonistreagents for use in this assay, the invention makes possible the priordetermination of the presence or absence of the side effect ofreactivity with the C140 receptor in candidate pharmaceuticals. Thisside effect will usually be undesired as it is believed that the C140receptor responds to enzymes such as serine proteases associated withtrauma and immune disturbances.

DISCLOSURE OF THE INVENTION

[0004] The invention provides methods and materials useful in assaysystems to determine the propensity of candidate pharmaceuticals toexert undesirable side effects. The isolation, recombinant productionand characterization of the C140 receptor permits the design of assaysystems using the receptor as a substrate and using agonists andantagonists for the receptor as control reagents in the assay.

[0005] Thus, in one aspect, the invention is directed to recombinantmaterials associated with the production of C140 receptor. Theseinclude, for example, transfected cells which can be cultured so as todisplay the C140 receptor on their surfaces, and thus provide an assaysystem for the interaction of materials with the native C140 receptor.In general, the limitations on the host cells useful in these assaysystems are that the cells have the appropriate mechanism to display thereceptor on their surfaces and contain the G-protein as mediator to theintracellular response. (However assays which merely assess binding donot require the G-protein.) Most animal cells meet these requirements.

[0006] In another aspect, the invention is directed to C140 receptoragonists which mimic the activated form of the extracellular portion ofthe receptor protein. These agonists are useful as control reagents inthe above-mentioned assays to verify the workability of the assaysystem. In addition, agonists for the C140 receptor may exhibithypotensive effects in vivo. Accordingly, the agonists may be also,themselves, useful as antihypertensives.

[0007] In still another aspect, the invention is directed to C140receptor antagonists. These antagonists comprise modified forms of theC140 receptor agonist peptides that lack the essential features requiredfor activation of the receptor. These antagonists bind to receptor, donot activate it, and prevent receptor activation by agonists and thenative receptor-binding ligand.

[0008] A second group of antagonists includes antibodies designed tobind specific portions of the receptor protein. In general, these aremonoclonal antibody preparations which are highly specific for anydesired region of the C140 receptor. The antibodies of the invention arealso useful in immunoassays for the receptor protein, for example, inassessing successful expression of the gene in recombinant systems.

[0009] Another aspect of the invention is to provide nucleic acidsencoding such a C140 receptor polypeptide and to use this nucleic acidto produce the polypeptide in recombinant cell culture for diagnosticuse or for potential therapeutic use in hemostatic or immune responseregulation.

[0010] In still other aspects, the invention provides an isolatednucleic acid molecule encoding a C140 receptor, labeled or unlabeled,and a nucleic acid sequence that is complementary to, or hybridizesunder stringent conditions to, a nucleic acid sequence encoding a C140receptor. The isolated nucleic acid molecule of the present inventionexcludes nucleic acid sequences which encode, or are complementary tonucleic acid sequences encoding, other known G protein-coupled receptorswhich are not C140 receptors, such as adrenergic receptors, neuropeptidereceptors, thrombin receptors, and the like.

[0011] In addition, the invention provides a replicable vectorcomprising a nucleic acid molecule encoding a C140 receptor operablylinked to control sequences recognized by a host transformed by thevector; host cells transformed with the vector; and a method of using anucleic acid molecule encoding a C140 receptor to effect the productionof a C140 receptor, comprising expressing the nucleic acid molecule in aculture of the transformed host cells and recovering a C140 receptorfrom the host cell culture. The nucleic acid sequence is also useful inhybridization assays for C140 receptor-encoding nucleic acid molecules.

[0012] In still further embodiments, the invention provides a method forproducing C140 receptors comprising inserting into the DNA of a cellcontaining the nucleic acid sequence encoding a C140 receptor atranscription modulatory element in sufficient proximity and orientationto the C140 receptor coding sequence to influence transcription thereof,with an optional further step comprising culturing the cell containingthe transcription modulatory element and the C140 receptor-encodingnucleic acid sequence.

[0013] In still further embodiments, the invention provides a cellcomprising a nucleic acid sequence encoding a C140 receptor and anexogenous transcription modulatory element in sufficient proximity andorientation to the above coding sequence to influence transcriptionthereof; and a host cell containing the nucleic acid sequence encoding aC140 receptor operably linked to exogenous control sequences recognizedby the host cell.

[0014] Still further is provided a method for obtaining cells havingincreased or decreased transcription of the nucleic acid moleculeencoding a C140 receptor, comprising:

[0015] (a) providing cells containing the nucleic acid molecule;

[0016] (b) introducing into the cells a transcription modulatingelement; and

[0017] (c) screening the cells for a cell in which the transcription ofthe nucleic acid molecule is increased or decreased.

[0018] In another aspect, the invention is related to assay systemswhich utilize recombinant C140 receptor to screen for agonist andantagonist activity of candidate drugs. This assay is especially usefulin assuring that these therapeutic agents do not have undesired sideeffects caused by activation or inhibition of the C140 receptor. In somecases agonist activity at this receptor system may have therapeuticutility. Some of these assay systems include the use of the agonistpeptides as positive controls. The assay can also be used to screen forantagonists which inhibit the agonistic effect.

[0019] Another aspect of the invention relates to the diagnosis ofconditions characterized by activation of the C140 receptor by detectionin fluids, such as blood or urine, of the peptide cleaved from the C140receptor when the receptor is activated. Another diagnostic methodincluded in the invention is visualization of the activated forms ofreceptor by localizing an imaging agent to activated receptor in situusing antibodies specific to the activated receptor.

[0020] Yet another aspect of this invention relates to the therapeutic,prophylactic and research uses of various techniques to block ormodulate the expression of a C140 receptor by interfering with thetranscription of translation of a DNA or RNA molecule encoding the C140receptor. This includes a method to inhibit or regulate expression ofC140 receptors in a cell comprising providing to the cell anoligonucleotide molecule which is antisense to, or forms a triple helixwith, C140 receptor-encoding DNA or with DNA regulating expression ofC140 receptor-encoding DNA, in an amount sufficient to inhibit orregulate expression of the C140 receptors, thereby inhibiting orregulating their expression. Also included is a method to inhibit orregulate expression of C140 receptors in a subject, comprisingadministering to the subject an oligonucleotide molecule which isantisense to, or forms a triple helix with, C140 receptor-encoding DNAor with DNA regulating expression of C140 receptor-encoding DNA, in anamount sufficient to inhibit or regulate expression of the C140receptors in the subject, thereby inhibiting or regulating theirexpression. The antisense molecule or triple helix-forming molecule inthe above methods is preferably a DNA or RNA oligonucleotide.

[0021] Additional aspects of the invention are directed topharmaceutical compositions containing the agonists and antagonists ofthe invention. The agonists of the invention are antihypertensives;conversely, the antagonists can elevate blood pressure if desired. Otheraspects of the invention include a pharmaceutical composition useful forinhibiting or regulating C140 receptor expression in a cell or in asubject at the level of transcription or translation, which compositioncomprises an antisense or triple helix-forming molecule as describedabove which corresponds to a portion of the sequence of the C140receptor-coding nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1A-1B show the DNA and deduced amino acid sequence ofmurine C140 receptor.

[0023] FIGS. 2A-2B show the DNA and deduced amino acid sequence of humanC140 receptor.

[0024]FIG. 3 shows a comparison of amino acid sequences for the humanC140 receptor and murine C140 receptor.

[0025]FIG. 4 shows a proposed model of C140 receptor activation based onthe deduced amino acid sequence.

[0026]FIG. 5 shows a comparison of amino acid sequences for the mouseC140 receptor and the human thrombin receptor.

[0027]FIG. 6 shows the results of Northern Blot to detect the presenceof mRNA encoding C140 receptor in various mouse tissues.

[0028]FIG. 7 shows a trace of blood pressure demonstrating the in vivohypotensive effect of a C140 agonist peptide.

[0029]FIGS. 8a-8 c show blood vessel dilation in rat femoral veininduced by a C140 receptor agonist peptide.

[0030]FIG. 8a shows these results in the immobilized vein;

[0031]FIG. 8b shows these results for the immobilized vein depleted ofendothelial cells.

[0032]FIGS. 9a-9 c show the results of an assay for activation of theC140 receptor, expressed in frog oocytes, by plasmin, kallikrein, ortrypsin. FIG. 9a shows the results for plasmin; FIG. 9b shows theresults for kallikrein; FIG. 9c shows the results for trypsin.

[0033] FIGS. 10A-10B show the nucleotide sequence and deduced amino acidsequence of a cDNA clone encoding murine C140 receptor.

[0034] FIGS. 11A-11B show the nucleotide sequence and deduced amino acidsequence of a cDNA clone encoding human C140 receptor.

[0035]FIG. 12 shows the results of in situ hybridization of a sectionednewborn mouse with mouse C140 receptor probes.

[0036]FIG. 13 shows a Northern blot of total RNA from human cell lineshybridized to a human C140 receptor probe.

MODES OF CARRYING OUT THE INVENTION

[0037] The characteristics of the C140 receptor elucidated by theinvention herein are summarized in FIGS. 1A/1B-4. FIGS. 1A-1B shows thecomplete DNA sequence of the clone encoding the murine receptor, alongwith the deduced amino acid sequence. As used herein, the “C140receptor” refers to receptor in any animal species corresponding to themurine receptor contained in clone C140 described in Example 1 herein.Using the native DNA encoding the murine form of this receptor, thecorresponding receptors in other species, including humans, asillustrated herein, may be obtained. FIGS. 2A-2B shows the correspondingDNA and deduced amino acid sequence of the human receptor.

[0038] The entire amino acid sequence of the murine receptor contains395 amino acids, including a 27 amino acid signal peptide which, whencleaved, results in a 368 amino acid mature receptor protein. Similarly,the human receptor is encoded by an open reading frame corresponding to398 amino acids including a probable 29 amino acid signal peptidesequence resulting in a 369 amino acid mature receptor protein, as shownin FIGS. 2A-2B.

[0039]FIG. 3 shows a comparison of the human and murine amino acidsequences; as shown, these sequences exhibit a high degree of homology.

[0040] Hydrophobicity/hydrophilicity plots of the sequences shown inFIGS. 1A-1B and 2A-2B indicate that the mature C140 receptor is a memberof the 7-transmembrane domain receptor family whose effect on the cellis mediated by G-protein. The mature C140 receptor has a relatively longextracellular amino acid extension containing several consensus sitesfor asparagine-linked glycosylation. It also contains a conservedasparagine in the first transmembrane region, the motif Leu-Ala-X-X-Aspin the second transmembrane region, a Trp in the fourth transmembraneregion and a carboxy terminal tail which contains multiple serine andthreonine residues. A proposed model of the in situ receptor is shown inFIG. 4.

[0041] Referring to FIG. 5, similarities to the thrombin receptor arereadily seen. FIG. 5 compares the amino acid sequence of murine C140with that of thrombin receptor. It is known that the thrombin receptoris activated by proteolytic cleavage of the Arg-Ser bond at positions 41and 42, which releases an activation peptide that permits refolding ofthe receptor and activation via the newly created amino terminus. In ananalogous manner, the C140 receptor is activated by cleavage of theArg-Ser bond at positions 34 and 35, also liberating an activationpeptide extending from position 1 of the putative mature protein to thecleavage site. It is believed that Arg-28 is the amino terminal aminoacid residue of the mature protein, so the activation peptide has thesequence RNNSKGR. This peptide could thus be used as an index foractivation of C140 receptor. In any event, the precise location of theN-terminus of the mature protein is unimportant for the design ofagonists or antagonists. The activation peptide is likely to be freelyfiltered by the kidney and possibly concentrated in the urine and can beused as an index to activation of the C140 receptor.

[0042] Release of the activation peptide permits refolding of thereceptor protein to activate the receptor. This is shown schematicallyin FIG. 4, which also shows that the conformational changes resultingfrom the liberation of the activation peptide and refolding results inan intracellular conformational change of the receptor. This hypothesisis confirmed by the finding that the C140 receptor can be activated by apeptide mimicking the new amino terminus created by the activation.Accordingly, mimics of the N-terminus of the new amino terminus on theactivated receptor behave as agonists therefor. The importance of thefirst five amino acids in the newly created amino terminus in thereceptor for receptor activation has also been confirmed hereinbelow.

[0043] Based on this information, and by analogy with the mechanismsunderlying trypsinogen activation to trypsin and activation of thethrombin receptor, it appears that the positively charged amino group onserine that is newly exposed when the ligand cleaves the receptor playsan important role in receptor activation. Peptides based on the agonistpeptide sequence that bind the C140 receptor, but which are modified tobe lacking the free α-amino group can function as antagonists of thisreceptor. Thus, modifications of the agonist peptides which lack thecapacity for specific activating interaction serve as C140 receptorantagonists.

[0044] Ordinarily, the C140 receptors and analogs thereof claimed hereinwill have an amino acid sequence having at least 75% amino acid sequenceidentity with a “common” C140 receptor sequence (such as that disclosedin FIGS. 1A-1B or FIGS. 2A-2B), more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to a common sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the known C140 receptor, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. None of N-terminal, C-terminal or internalextensions, deletions, or insertions into the C140 receptor sequenceshall be construed as affecting homology.

[0045] Thus, the claimed C140 receptor and analog molecules that are thesubject of this invention include molecules having the C140 receptoramino acid sequence; fragments thereof having a consecutive sequence ofat least 10, 15, 20, 25, 30 or 40 amino acid residues from a common C140receptor sequence; amino acid sequence variants of a common C140receptor sequence wherein an amino acid residue has been inserted N- orC-terminal to, or within, the C140 receptor sequence or its fragments asdefined above; amino acid sequence variants of the common C140 receptorsequence or its fragment as defined above which have been substituted byanother residue. C140 receptor polypeptides include those containingpredetermined mutations by, e.g., homologous recombination,site-directed or PCR mutagenesis, and C140 receptor polypeptides ofother animal species, including but not limited to rabbit, rat, murine,porcine, bovine, ovine, equine and non-human primate species, andalleles or other naturally occurring variants of the C140 receptor ofthe foregoing species and of human sequences; derivatives of thecommonly known C140 receptor or its fragments wherein the C140 receptoror its fragments have been covalently modified by substitution,chemical, enzymatic, or other appropriate means with a moiety other thana naturally occurring amino acid (for example a detectable moiety suchas an enzyme or radioisotope); glycosylation variants of C140 receptor(insertion of a glycosylation site or deletion of any glycosylation siteby deletion, insertion or substitution of appropriate amino acid); andsoluble forms of C140.

[0046] The novel proteins and peptides of the present invention arepreferably those which share a common biological activity with the C140receptor, including but not limited to an effector or receptor functionor cross-reactive antigenicity. Such fragments and variants exclude anyC140 receptor polypeptide heretofore made public, including any knownprotein or polypeptide of any animal species, which is otherwiseanticipatory under 35 U.S.C. §102 as well as polypeptides obvious oversuch known protein or polypeptides under 35 U.S.C. §103. Specifically,the present C140 receptor proteins, analogs, fragments and variantsexclude other known G protein-coupled receptors which are not C140receptors, such as adrenergic receptors, neuropeptide receptors,thrombin receptors, and the like.

COMPOUNDS OF THE INVENTION

[0047] The nomenclature used to describe the peptide compounds of theinvention follows the conventional practice where the N-terminal aminogroup is assumed to be to the left and the carboxy group to the right ofeach amino acid residue in the peptide. In the formulas representingselected specific embodiments of the present invention, the amino- andcarboxy-terminal groups, although often not specifically shown, will beunderstood to be in the form they would assume at physiological pHvalues, unless otherwise specified. Thus, the N-terminal H⁺ ₂ andC-terminal O⁻ at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

[0048] In the peptides shown, each gene-encoded residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the amino acid, in accordance withthe following conventional list: One-Letter Three-letter Amino AcidSymbol Symbol Alanine A Ala Arginine R Arg Asparagine N Asn Asparticacid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine GGly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K LysMethionine M Met Phenylalanine F Phe Proline P Pro Serine S SerThreonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

[0049] The amino acids not encoded genetically are abbreviated asindicated in the discussion below.

[0050] In the specific peptides shown in the present application, theL-form of any amino acid residue having an optical isomer is intendedunless the D-form is expressly indicated by a dagger superscript (†).

[0051] The compounds of the invention are peptides which are partiallydefined in terms of amino acid residues of designated classes. Aminoacid residues can be generally subclassified into four major subclassesas follows:

[0052] Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

[0053] Basic: The residue has a positive charge due to association withH ion at physiological pH and the residue is attracted by aqueoussolution so as to seek the surface positions in the conformation of apeptide in which it is contained when the peptide is in aqueous mediumat physiological pH.

[0054] Neutral/nonpolar: The residues are not charged at physiologicalpH and the residue is repelled by aqueous solution so as to seek theinner positions in the conformation of a peptide in which it iscontained when the peptide is in aqueous medium. These residues are alsodesignated “hydrophobic” herein.

[0055] Neutral/polar: The residues are not charged at physiological pH,but the residue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

[0056] It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged,” a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

[0057] Amino acid residues can be further subclassified as cyclic ornoncyclic, and aromatic or nonaromatic, self-explanatory classificationswith respect to the side chain substituent groups of the residues, andas small or large. The residue is considered small if it contains atotal of 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always nonaromatic.

[0058] For the naturally occurring protein amino acids,subclassification according to the foregoing scheme is as follows.

[0059] Acidic: Aspartic acid and Glutamic acid;

[0060] Basic/noncyclic: Arginine, Lysine;

[0061] Basic/cyclic: Histidine;

[0062] Neutral/polar/small: Glycine, serine, cysteine;

[0063] Neutral/nonpolar/small: Alanine;

[0064] Neutral/polar/large/nonaromatic: Threonine, Asparagine,Glutamine;

[0065] Neutral/polar/large aromatic: Tyrosine;

[0066] Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine, Leucine,Methionine;

[0067] Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan

[0068] The gene-encoded secondary amino acid proline, althoughtechnically within the group neutral/nonpolar/large/cyclic andnonaromatic, is a special case due to its known effects on the secondaryconformation of peptide chains, and is not, therefore, included in thisdefined group.

[0069] Certain commonly encountered amino acids, which are not encodedby the genetic code, include, for example, beta-alanine (beta-Ala), orother omega-amino acids, such as 3-amino propionic, 2,3-diaminopropionic (2,3-diaP), 4-amino butyric and so forth, alpha-aminisobutyricacid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit),t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine(N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine(Nle), cysteic acid (Cya) 2-naphthylalanine (2-Nal);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); and methionine sulfoxide (MSO). These alsofall conveniently into particular categories.

[0070] Based on the above definitions,

[0071] Sar, beta-Ala, 2,3-diaP and Aib are neutral/nonpolar/small;

[0072] t-BuA, t-BuG, N-MeIle, Nle, Mvl and Cha areneutral/nonpolar/large/nonaromatic;

[0073] Orn is basic/noncyclic;

[0074] Cya is acidic;

[0075] Cit, Acetyl Lys, and MSO are neutral/polar/large/nonaromatic; and

[0076] Phg, Nal, Thi and Tic are neutral/nonpolar/large/aromatic.

[0077] The various omega-amino acids are classified according to size asneutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,4-aminobutyric) or large (all others).

[0078] Other amino acid substitutions of those encoded in the gene canalso be included in peptide compounds within the scope of the inventionand can be classified within this general scheme according to theirstructure.

[0079] All of the compounds of the invention, when an amino acid formsthe C-terminus, may be in the form of the pharmaceutically acceptablesalts or esters. Salts may be, for example, Na⁺, K⁺, Ca⁺², Mg⁺² and thelike; the esters are generally those of alcohols of 1-6C.

[0080] In all of the peptides of the invention, one or more amidelinkages (—CO—NH—) may optionally be replaced with another linkage whichis an isostere such as —CH₂NH—, —CH₂S—, —CH₂CH₂, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be madeby methods known in the art. The following references describepreparation of peptide analogs which include these alternative-linkingmoieties: Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,“Peptide Backbone Modifications” (general review); Spatola, A. F., in“Chemistry and Biochemistry of Amino Acids Peptides and Proteins,” B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (generalreview); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (generalreview); Hudson, D., et al., Int J Pept Prot Res (1979) 14:177-185(—CH₂NH—, —CH₂CH₂—); Spatola, A. F., et al., Life Sci (1986)38:1243-1249 (—CH₂—S); Hann, M. M., J Chem Soc Perkin Trans I (1982)307-314 (—CH—CH—, cis and trans); Almquist, R. G., et al., J Med Chem(1980) 23:1392-1398 (—COCH₂—); Jennings-White, C., et al., TetrahedronLett (1982) 23:2533 (—COCH₂—); Szelke, M., et al., European ApplicationEP 45665 (1982) CA:97:39405 (1982) (—CH(OH)CH₂—); Holladay, M. W., etal., Tetrahedron Lett (1983) 24:4401-4404 (—C(OH)CH₂—); and Hruby, V.J., Life Sci (1982) 31:189-199 (—CH₂—S—)

[0081] A. Agonists

[0082] The agonists of the invention comprise a series of peptides ofthe formula

AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-Z   (1)

[0083] wherein AA₁ is a small amino acid or threonine;

[0084] AA₂ and AA₃ are each independentlyneutral/nonpolar/large/nonaromatic amino acids;

[0085] AA₄ is a small amino acid;

[0086] AA₅ is a basic amino acid;

[0087] AA₆ may be present or absent and, if present, is aneutral/nonpolar/large/nonaromatic amino acid;

[0088] AA₇ is absent if AA₆ is absent and may be present or absent ifAA₆ is present, and is an acidic amino acid; and

[0089] Z is a substituent that does not interfere with agonist activity.

[0090] The peptide of formula 1 can be extended (shown as included in Z)at the C-terminus (but not the N-terminus) by further amino acidsequence to comprise a noninterfering substituent.

[0091] At the C-terminus of the compounds of formula 1, the carboxylgroup may be in the underivatized form or may be amidated or may be anester; in the underivatized form the carboxyl may be as a free acid or asalt, preferably a pharmaceutically acceptable salt.

[0092] If the C-terminus is amidated, the nitrogen atom of the amidogroup, covalently bound to the carbonyl carbon at the C-terminus, willbe NR′R′, wherein each R′ is independently hydrogen or is a straight orbranched chain alkyl of 1-6C, such alkyls are 1-6C straight- orbranched-chain saturated hydrocarbyl residues, such as methyl, ethyl,isopentyl, n-hexyl, and the like. Representatives of such amido groupsare: —NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃, —NHCH₂CH(CH₃)₂, and—NHCH₂CH(CH₃)CH₂CH₃, among others. Furthermore, either or both R′ may inturn optionally be substituted by one or more substituents such as, forexample, —OR′, —NR′R′, halo, —NR′CNR′NR′R′ and the like, wherein each R′is as independently defined above. Thus, Z may be —OH, or an ester (OR′)or salt forms thereof, or —NR′R′ wherein R′ is as above defined.

[0093] Preferred embodiments of AA₁ are Ser on 2,3-diaminopropionyl(2,3-diaP). Preferred embodiments of AA₂ and AA₃ are Val, Ile, Cha andLeu. Preferred embodiments for the residues in the remainder of thecompound of formula (1) are those wherein AA₄ is Gly, AA₅ is Lys, Arg orHar, AA₆, if present, is Val, Ile, Cha or Leu, and AA₇, if present, isAsp or Glu. Particularly preferred are compounds of formula (1) whichare selected from the group consisting of SLIGRLETQPPIT, SLIGRLETQPPI,SLIGRLETQPP, SLIGRLETQP, SLIGRLETQ, SLIGRLET, SLIGRLE, SLIGRL, SLIGR,SLLGKVDGTSHVT, SLLGKVDGTSHV, SLLGKVDGTSH, SLLGKVDGTS, SLLGKVDGT,SLLGKVDG, SLLGKVD, SLLGKV, SLLGK, S(Cha)IGR, S(Cha)LGK, (2,3-diaP)-IGR,(2,3-diaP)LLGK, SLLGKR-NH₂, SLIGRR-NH₂, S(Cha)LGKK-NH₂, S(Cha)IGRK-NH₂,(2,3-diaP)-LIGRK-NH₂, (2,3-diaP)-LLGKK-NH₂ and the amidated formsthereof.

[0094] B. Antagonists

[0095] Compounds of the invention which interfere with activitiesmediated by the C140 receptor include modified agonist peptides lackingthe N-terminal serine residue; and antibodies which are immunoreactivewith various critical positions on the C140 receptor.

[0096] Peptide Antagonists

[0097] The antagonists of the first group—modified agonists—can berepresented by the formula:

X-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-Z

[0098] wherein X is an amino acid residue other than ser, ala, thr, cys,2,3-diaP or gly or is a desamino or alkylated or acylated amino acid,

[0099] wherein AA₂ and AA₃ are each independentlyneutral/nonpolar/large/nonaromatic amino acids;

[0100] AA₄ is a small amino acid;

[0101] AA₅ is a basic amino acid;

[0102] AA₆ may be present or absent and, if present, is aneutral/nonpolar/large/nonaromatic amino acid;

[0103] AA₇ is absent if AA₆ is absent and may be present or absent ifAA₆ is present, and is an acidic amino acid; and

[0104] Z is a substituent that does not interfere with agonist activity.

[0105] Preferred acyl groups are of the formula RCO— wherein Rrepresents a straight or branched chain alkyl of 1-6C. Acetyl isparticularly preferred.

[0106] Preferred embodiments of X include residues of3-mercaptopropionic acid (Mpr), 3-mercaptovaleric acid (Mvl),2-mercaptobenzoic acid (Mba) and S-methyl-3-mercaptopropionic acid(SMeMpr). Preferred embodiments for AA₂ through AA₇ are as described forthe agonists above; Z is also as thus described.

[0107] Particularly preferred among the antagonist peptides of thisclass are those selected from the group consisting of Mpr-LLGK,Mpr-LIGR, Mpr-(Cha)LKG, Mpr-(Cha)IGR, Mpr-LLGKK-NH₂, Mpr-LIGRK-NH₂,Mpr-LIGRKETQP-NH₂, Mpr-LLGKKDGTS-NH₂,(n-pentyl)₂-N-Leu-Ile-Gly-Arg-Lys-NH₂ and(Me-N-(n-pentyl)-Leu-Ile-Gly-Arg-Lys-NH₂.

[0108] Antibodies

[0109] Antagonists which are antibodies immunoreactive with criticalpositions of the C140 receptor are obtained by immunization of suitablemammalian subjects with peptides containing as antigenic regions thoseportions of the C140 receptor intended to be targeted by the antibodies.Critical regions include the region of proteolytic cleavage, the segmentof the extracellular segment critical for activation (this includes thecleavage site), and the portions of the sequence which form theextracellular loops, in particular, that region which interacts with theN-terminus of the activated receptor extracellular region. The agonistpeptides of the invention may be used as immunogens in this case.

[0110] Thus, peptides which contain the proteolytic region, namely, forexample, SKGRSLIGRLET, the extracellular loops, such as those includingISY HLHGNNWVYGEALC; QTIYIPALNITTCHDVLPEEVLVGDMFNYFL; andHYFLIKTQRQSHVYA. The agonist peptides described below are also useful asimmunogens.

[0111] The antibodies are prepared by immunizing suitable mammalianhosts in appropriate immunization protocols using the peptide haptensalone, if they are of sufficient length, or, if desired, or if requiredto enhance immunogenicity, conjugated to suitable carriers. Methods forpreparing immunogenic conjugates with carriers such as BSA, KLH, orother carrier proteins are well known in the art. In some circumstances,direct conjugation using, for example, carbodiimide reagents may beeffective; in other instances linking reagents such as those supplied byPierce Chemical Co., Rockford, Ill., may be desirable to provideaccessibility to the hapten. The hapten peptides can be extended at theamino or carboxy terminus with a Cys residue or interspersed withcysteine residues, for example, to facilitate linking to carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

[0112] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions, useof monoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the peptide hapten or isthe C140 receptor itself displayed on a recombinant host cell. When theappropriate immortalized cell culture secreting the desired antibody isidentified, the cells can be cultured either in vitro or by productionin ascites fluid.

[0113] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

[0114] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of receptor can also be produced in the context ofchimeras with multiple species origin.

[0115] The antibodies thus produced are useful not only as potentialantagonists for the receptor, filling the role of antagonist in theassays of the invention, but are also useful in immunoassays fordetecting the activated receptor. As such these antibodies can becoupled to imaging agents for administration to a subject to allowdetection of localized antibody to ascertain the position of C140receptors in either activated or unactivated form. In addition, thesereagents are useful in vitro to detect, for example, the successfulproduction of the C140 receptor deployed at the surface of therecombinant host cells.

[0116] Preparation of Peptide Agonists and Antagonists

[0117] The peptide agonists and antagonists of the invention can beprepared using standard solid phase (or solution phase) peptidesynthesis methods, as is known in the art. In addition, the DNA encodingthese peptides may be synthesized using commercially availableoligonucleotide synthesis instrumentation and produced recombinantlyusing standard recombinant production systems. The production usingsolid phase peptide synthesis is necessitated if non-gene-encoded aminoacids are to be included.

[0118] Preparation of C140 Receptor Nucleic Acids

[0119] C140 receptor “nucleic acid” is defined as RNA or DNA thatencodes a C140 receptor, or is complementary to nucleic acid sequenceencoding a C140 receptor, or hybridizes to such nucleic acid and remainsstably bound to it under stringent conditions, or encodes a polypeptidesharing at least 75% sequence identity, preferably at least 80%, andmore preferably at least 85%, with the translated amino acid sequencesshown in FIGS. 3, 10A-10B or 11A-11B. It is typically at least about 10nucleotides in length and preferably has C140 receptor biological orimmunological activity, including the nucleic acid encoding anactivation peptide fragment having the nucleotide sequence shown in FIG.4. Specifically contemplated are genomic DNA, cDNA, mRNA and antisensemolecules, as well as nucleic acids based on alternative backbone orincluding alternative bases whether derived from natural sources orsynthesized. Such hybridizing or complementary nucleic acid, however, isdefined further as being novel and unobvious over any prior art nucleicacid including that which encodes, hybridizes under stringentconditions, or is complementary to nucleic acid encoding a known Gprotein-coupled receptor.

[0120] “Stringent conditions” are those that (1) employ low ionicstrength and high temperature for washing, for example, 0,015MNaCl/0.0015M sodium titrate/0.1% NaDodSO4 at 50° C., or (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 mu g/ml), 0.1% SDS, and 10% dextransulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

[0121] “Isolated” nucleic acid will be nucleic acid that is identifiedand separated from contaminant nucleic acid encoding other polypeptidesfrom the source of nucleic acid. The nucleic acid may be labeled fordiagnostic and probe purposes, using any label known and described inthe art as useful in connection with diagnostic assays.

[0122] Of particular interest is a C140 receptor nucleic acid thatencodes a full-length molecule, including but not necessarily the nativesignal sequence thereof. Nucleic acid encoding full-length protein isobtained by screening selected cDNA (not kidney) or genomic librariesusing the deduced amino acid sequence disclosed herein for the firsttime, and, if necessary, using conventional primer extension proceduresto secure DNA that is complete at its 5′ coding end. Such a clone isreadily identified by the presence of a start codon in reading framewith the original sequence.

[0123] DNA encoding an amino acid sequence variant of a C140 receptor isprepared as described below or by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of a C140 receptor.

[0124] Techniques for isolating and manipulating nucleic acids aredisclosed for example by the following documents: U.S. Pat. No.5,030,576, U.S. Pat. No. 5,030,576 and International Patent PublicationsWO94/11504 and WO93/03162. See, also, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1989, and Ausubel, F. M. et al. CurrentProtocols in Molecular Biology, Vol. 2, Wiley—Interscience, New York,1987. Disclosures of these documents are expressly incorporated hereinby reference in their entireties.

[0125] Recombinant Production of C140 Receptor for Use in Assays

[0126] The invention provides recombinant materials for the productionof C140 receptor for display on the surface of recombinant cells.Production of the receptor using these recombinant methods provides auseful reagent to determine the ability of a candidate drug to bind to,to activate, or to antagonize the C140 receptor. Determination of theseproperties is essential in evaluating the specificity of drugs intendedfor binding other related receptors.

[0127] For this recombinant production, a DNA sequence encoding the C140receptor, such as those set forth in FIGS. 1A-1B and 2A-2B, or theirsubstantial equivalents or their degenerate analogs, is prepared eitherby retrieval of the native sequence, as set forth below, or by usingsubstantial portions of the known native sequence as probe, or can besynthesized de novo using standard procedures. The DNA is ligated intoexpression vectors suitable for the desired host and transformed intocompatible cells. The cells are cultured under conditions which favorthe expression of the C140 receptor encoding gene and the cellsdisplaying the receptor on the surface are harvested for use in theassays.

[0128] The host cells are typically animal cells, most typicallymammalian cells. In order to be useful in the assays, the cells musthave intracellular mechanisms which permit the receptor to be displayedon the cell surface in the configuration shown generally in FIG. 4herein. If the assay uses cellular response to activated receptor as adetection system, the cells must also contain a G-protein linkedmechanism for response to activation of the receptors. Most mammalianand other animal cells fulfill these qualifications.

[0129] Particularly useful cells for use in the method of the inventionare Xenopus laevis frog oocytes, which typically utilize cRNA ratherthan standard recombinant expression systems proceeding from the DNAencoding the desired protein. Capped RNA (at the 5′ end) is typicallyproduced from linearized vectors containing DNA sequences encoding thereceptor. The reaction is conducted using RNA polymerase and standardreagents. cRNA is recovered, typically using phenol/chloroformprecipitation with ethanol and injected into the oocytes.

[0130] The animal host cells expressing the DNA encoding the C140receptor or the cRNA-injected oocytes are then cultured to effect theexpression of the encoding nucleic acids so as to produce the C140receptor displayed in a manner analogous to that shown in FIG. 4 ontheir surfaces. These cells then are used directly in assays forassessment of a candidate drug to bind, antagonize, or activate thereceptor.

[0131] Assays

[0132] In one type of easily conducted assay, competition of thecandidate drug for binding to the receptor with either agonist or knownbinding antagonist can be tested. In one method, the competing agonistor antagonist may be labeled; the labeled substance known to bind thereceptor can, of course, be a synthetic peptide. In one typicalprotocol, varying concentrations of the candidate are supplied alongwith a constant concentration of labeled agonist or antagonist and theinhibition of a binding of label to the receptor can be evaluated usingknown techniques.

[0133] In a somewhat more sophisticated approach, the effect ofcandidate compounds on agonist-induced responses can be measured in thecells recombinantly expressing the C140 receptor as described below.Assay systems for the effect of activation of receptor on these cellsinclude calcium mobilization and voltage clamp which are describedherein in further detail. These assays permit an assessment of theeffect of the candidate drug on the receptor activity rather than simplyability to bind to the receptor.

[0134] Agonist-induced increases in ⁴⁵Ca release by oocytes expressingcRNA encoding C140 receptor or other recombinant cells producing C140receptor are assessed by published techniques (Williams, J. A., et al.,Proc Natl Acad Sci USA (1988) 85:4939-4943). Briefly, intracellularcalcium pools are labeled by incubating groups of 30 oocytes in 300 μlcalcium-free modified Barth's solution (MBSH) containing 50 μCi ⁴⁵CaCl₂(10-40 mCi/mg Ca; Amersham) for 4 hours at RT. The labeled oocytes orcells are washed, then incubated in MBSH II without antibiotics for 90minutes. Groups of 5 oocytes are selected and placed in individual wellsin a 24-well tissue culture plate (Falcon 3047) containing 0.5 ml/wellMBSH II without antibiotics. This medium is removed and replaced withfresh medium every 10 minutes; the harvested medium is analyzed byscintillation counting to determine ⁴⁵Ca released by the oocytes duringeach 10-minute incubation. The 10-minute incubations are continued untila stable baseline of ⁴⁵Ca release per unit time is achieved. Twoadditional 10-minute collections are obtained, then test mediumincluding agonist is added and agonist-induced ⁴⁵Ca release determined.

[0135] Using the above assay, the ability of a candidate drug toactivate the receptor can be tested directly. In this case, the agonistsof the invention are used as controls. In addition, by using the agonistof the invention to activate the recombinant receptor, the effect of thecandidate drug on this activation can be tested directly. Recombinantcells expressing the nucleic acids encoding the receptor are incubatedin the assay in the presence of agonist with and without the candidatecompound. A diminution in activation in the presence of the candidatewill indicate an antagonist effect. Conversely, the ability of acandidate drug to reverse the antagonist effects of an antagonist of theinvention may also be tested.

[0136] In an alternative to measuring calcium mobilization, the voltageclamp assay can be used as a measure for receptor activation.Agonist-induced inward chloride currents are measured in voltage-clampedoocytes expressing C140 receptor encoding cRNA or cells expressing DNAfrom recombinant expressions systems essentially as previously described(Julius, D., et al, Science (1988) 241:558-563) except that the singleelectrode voltage-clamp technique is employed.

[0137] Detection of Activated Receptors

[0138] In one embodiment, the availability of the recombinant C140receptor protein permits production of antibodies which areimmunospecific to the activated form of the receptor which can then beused for diagnostic imaging of activated receptors in vivo. Theseantibodies are produced either to the activated form of the receptorproduced recombinantly, or to the peptide representing the “new aminoterminal” peptide described herein. The resulting antibodies, or theimmunospecific fragments thereof, such as the Fab, Fab′, Fab′₂ fragmentsare then conjugated to labels which are detected by known methods, suchas radiolabels including technetium⁹⁹ and indium¹¹¹ or other radioactivelabels as is known in the art. When injected in vivo, these antibodieshome to the sites of activated receptor, thus permitting localization ofareas containing activated receptors.

[0139] In another embodiment, the presence of the activation peptide inbody fluids or in culture media can be detected and measured. Antibodiesare made to the activation peptide as described above and can beemployed in standard ELISA or RIA assays to detect excess amounts of theactivation peptide in, for example, urine.

[0140] Administration of Agonists and Antagonists as Pharmaceuticals

[0141] The peptides of the invention which behave as agonists areadministered in conventional formulations for systemic administration asis known in the art. Typical such formulations may be found, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton Pa., latest edition.

[0142] Preferred forms of systemic administration of peptides includeinjection, typically-by intravenous injection. Other injection routes,such as subcutaneous, intramuscular, or intraperitoneal, can also beused. More recently, alternative means for systemic administration ofpeptides have been devised which include transmucosal and transdermaladministration using penetrants such as bile salts or fusidic acids orother detergents. In addition, if properly formulated in enteric orencapsulated formulations, oral administration may also be possible.Administration of these compounds may also be topical and/or localized,in the form of salves, pastes, gels and the like.

[0143] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe patient's condition, and the judgment of the attending physician.Suitable dosage ranges, however, are in the range of 0.1-100 μg/kg ofsubject. Wide variations in the needed dosage, however, are to beexpected in view of the variety of peptides available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization as is well understood in the art.

[0144] As shown hereinbelow, the agonists of the invention behave asantihypotensives; antagonists have the opposite effect. Thus, patientswhose blood pressure needs to be raised or lowered benefit by theadministration of the suitable peptide.

[0145] In addition, the agonists have anti-inflammatory and woundhealing properties.

[0146] Antisense, Triple Helix and Gene Therapy Aspects

[0147] The constitutive expression of antisense RNA in cells has beenshown to inhibit the expression of about 20 different genes in mammalsand plants, and the list continually grows (Hambor, J. E. et al., J.Exp. Med. 168:1237-1245 (1988); Holt, J. T. et al., Proc. Nat. Acad.Sci. 83:4794-4798 (1986); Izant, J. G. et al., Cell 36:1007-1015 (1984);Izant, J. G., et al., Science 229:345-352 (1985) and De Benedetti, A. etal., Proc. Nat. Acad. Sci. 84:658-662 (1987)). Possible mechanisms forthe antisense effect are the blockage of translation or prevention ofsplicing, both of which have been observed in vitro. Interference withsplicing allows the use of intron sequences (Munroe, S. H., EMBO. J.7:2523-2532 (1988) which should be less conserved and therefore resultin greater specificity in inhibiting expression of a protein of onespecies but not its homologue in another species.

[0148] Therapeutic gene regulation is accomplished using the “antisense”approach, in which the function of a target gene in a cell or organismis blocked, by transfection of DNA, preferably an oligonucleotide,encoding antisense RNA which acts specifically to inhibit expression ofthe particular target gene. The sequence of the antisense DNA isdesigned to result in a full or preferably partial antisense RNAtranscript which is substantially complementary to a segment of the geneor mRNA which it is intended to inhibit. The complementarity must besufficient so that the antisense RNA can hybridize to the target gene(or mRNA) and inhibit the target gene's function, regardless of whetherthe action is at the level of splicing, transcription or translation.The degree of inhibition, readily discernible by one of ordinary skillin the art without undue experimentation, must be sufficient to inhibit,or render the cell incapable of expressing, the target gene. One ofordinary skill in the art will recognize that the antisense RNA approachis but one of a number of known mechanisms which can be employed toblock specific gene expression.

[0149] By the term “antisense” is intended an RNA sequence, as well as aDNA sequence coding therefor, which is sufficiently complementary to aparticular mRNA molecule for which the antisense RNA is specific tocause molecular hybridization between the antisense RNA and the mRNAsuch that translation of the mRNA is inhibited. Such hybridization mustoccur under in vivo conditions, that is, inside the cell. The action ofthe antisense RNA results in specific inhibition of gene expression inthe cell. (See: Albers, B. et al., MOLECULAR BIOLOGY OF THE CELL, 2ndEd., Garland Publishing, Inc., New York, N.Y. (1989), in particular,pages 195-196.

[0150] The antisense RNA of the present invention may be hybridizable toany of several portions of a target mRNA, including the coding sequence,a 3′ or 5′ untranslated region, or other intronic sequences. A preferredantisense RNA is that complementary to the human C140 receptor mRNA. Asis readily discernible by one of skill in the art, the minimal amount ofhomology required by the present invention is that sufficient to resultin hybridization to the specific target mRNA and inhibition of itstranslation or function while not affecting function of other mRNAmolecules and the expression of other genes.

[0151] Antisense RNA is delivered to a cell by transformation ortransfection with a vector into which has been placed DNA encoding theantisense RNA with the appropriate regulatory sequences, including apromoter, to result in expression of the antisense RNA in a host cell.

[0152] “Triple helix” or “triplex” approaches involve production ofsynthetic oligonucleotides which bind to the major groove of a duplexDNA to form a colinear triplex. Such triplex formation can regulate andinhibit cellular growth. See, for example: Hogan et al., U.S. Pat. No.5,176,996; Cohen, J. S. et al., Sci. Amer., Dec. 1994, p. 76-82; Helene,C., Anticancer Drug Design 6:569-584 (1991); Maher III, L. J. et al.,Antisense Res. Devel. 1:227-281 (Fall 1991); Crook, S. T. et al. eds.,ANTISENSE RESEARCH AND APPLICATIONS, CRC Press, 1993. It is based inpart on the discovery that a DNA oligonucleotide can bind by triplexformation to a duplex DNA target in a gene regulatory region, therebyrepressing transcription initiation (Cooney M. et. al. (1988) Science241:456). The present invention utilizes methods such as those of Hoganet al., supra (herein incorporated by reference in its entirety), todesigning oligonucleotides which will bind tightly and specifically to aduplex DNA target comprising part of the C140 receptor-encoding DNA or aregulatory sequence thereof. Such triplex oligonucleotides can thereforebe used as a class of drug molecules to selectively manipulate theexpression of this gene.

[0153] Thus the present invention is directed to providing to a cell oradministering to a subject a synthetic oligonucleotide in sufficientquantity for cellular uptake and binding to a DNA duplex of the targetC140 receptor-coding DNA sequence or a regulatory sequence thereof, suchthat the oligonucleotide binds to the DNA duplex to form a colineartriplex. This method is used to inhibit expression of the receptor oncells in vitro or in vivo. Preferably the target sequence is positionedwithin the DNA domain adjacent to the RNA transcription origin. Thismethod can also be used to inhibit growth of cells which is dependent onexpression of this receptor. The method may also be used to alter therelative amounts or proportions of the C140 receptor expressed on cellsor tissues by administering such a triplex-forming syntheticoligonucleotide.

[0154] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 Isolation of the Gene Encoding Murine C140 Receptor

[0155] A mouse cosmid genomic library (obtained from Dr. R. A. Wetsel,Washington University School of Medicine, St. Louis, Mo. and describedin Wetsel, R. A. et al., J Biol Chem (1990) 265:2435-2440) was screenedwith two ³²P-labeled oligonucleotides corresponding to bp 190-249 and742-801, respectively, of the bovine substance K receptor cDNA (Masu, Y.et al., Nature (1987) 329:836-838). The hybridization conditions are5×SSC, 5×Denhardt's, 0.1% SDS, 0.1 mg/ml sperm DNA, 10⁶ cpm/ml oflabeled oligonucleotides, 60° C. overnight, followed by washing with1×SSC, 0.1% SDS at 60° C.

[0156] In one of the clones isolated (C140) the hybridizing region waslocalized to a 3.7 kb PstI fragment. This fragment was subcloned intothe commercially available pBluescript vector. The hybridizing andadjacent regions were sequenced in both orientations by the Sanger chaintermination method. FIGS. 1A-1B shows both the nucleotide sequence andthe deduced amino acid sequence of the mouse C140 receptor. Thetentative signal sequence (SP) and the seven transmembrane regions areoverlined, potential asparagine-linked glycosylation sites are markedwith bold arrows, and the putative protease receptor cleavage site atArg34-Ser35 is marked with an open arrow.

EXAMPLE 2 Isolation of the Gene Encoding Human C140 Receptor

[0157] The availability of genomic DNA encoding the mouse protease C140receptor permitted the retrieval of the corresponding human gene. Ahuman genomic library cloned in the vector EMBL3 was screened at exactlythe conditions in Example 1 using the entire coding region of the murineclone as a probe. The recovered human gene including the DNA sequenceand the deduced amino acid sequence are shown in FIGS. 2A-2B. Subsequentexperiments indicated that the human C140 gene is located in the sameregion of the long arm of chromosome number 5 (5q12-5q13) as has beenreported for the human thrombin receptor gene.

[0158] In addition, a 1.1 kb genomic DNA fragment was obtained fromGenome Systems Inc., commercial screening service as was PCR-positivewith a primer pair that generates a fragment spanning 350-nucleotides ofthe human C140 protein coding region. A 1.1 kb bamH1 fragment wassubcloned and sequenced and found to contain 800-nucleotides of promotersequence. The promoter lacks both a TATA box and a CAAT box but is richin G's and C's; features common to promoters of many housekeeping genes.Two binding elements specific for SP1 and AP2 were identified.

EXAMPLE 3 Comparison of Related G-Protein Receptors

[0159] As shown in FIG. 3, the deduced amino acid sequence of the humanprotease C140 receptor shows extensive similarity (>90%) to the mousesequence.

[0160]FIG. 5 shows an amino acid sequence alignment between the mouseC140 receptor and the related G-protein receptor human thrombin receptor(Coughlin, S. Cell). The tentative signal sequences (SP), transmembraneregions, and protease cleavage sites are marked.

EXAMPLE 4 Recovery of Mouse C140 cDNA

[0161] A cDNA library from a mouse stomach was constructed in λ gt10 andscreened with a probe encompassing the C1040 genomic DNA. A single phageclone was isolated and cut with EcoRI. The insert was cloned intopBluescript and pSG5 and sequenced.

[0162] The isolated cDNA was 2732 nucleotides long including a 16 basepolyA-stretch; 5′ RACE resulted in the addition of only 27 bases to the5′ end. The 5′ end of the apparent coding region differs from the 5′ endof the open reading frame of genomic DNA; it is believed that the 5′ endof the cDNA is correct. The complete nucleotide sequence and deducedamino acid sequence of murine cDNA encoding C140 is shown in FIGS.10A-10B.

EXAMPLE 5 Recovery of Human cDNA Encoding C140

[0163] A human intestinal tumor cDNA library was subjected to PCR usingprimers designed from the genomic clone of Example 2 and the amplifiedfragment was cloned in pSG5 and sequenced. The nucleotide sequence anddeduced amino acid sequence are shown in FIGS. 11A-11B. There are fouramino acid differences between the cDNA encoded sequence and thatencoded by the genomic DNA as is shown in FIGS. 11A-11B.

EXAMPLE 6 Activation of Protease C140 Receptor in Oocytes

[0164] Both native and mutant C140 receptors were produced in oocytesand activated with a peptide mimicking the new amino-terminus”, or bythe proteolytic enzyme trypsin (which cleaves the extracellular region).Native receptors were produced by cloning the coding region of thereceptor gene, using the polymerase chain reaction, into the expressionvector pSG-5 (Green, S. et al., Nucleic Acid Res (1988) 16:369). Theorientation and integrity of the cloned coding region was verified bydetermining the nucleotide sequence with the Sanger chain-terminationmethod. Site-directed mutagenesis was employed to construct mutantreceptors in the pSG-5. Three mutant receptors were made, in whichserine-35 was replaced with proline, arginine, and histidine,respectively. The nucleotide sequences of the three mutants was verifiedas above.

[0165] In order to produce the receptor at the surface of oocytes, cRNAencoding the receptor was produced as follows. pSG-5 C140 plasmid DNAwas made linear by digestion with XbaI, and capped cRNA was produced invitro using T7 RNA polymerase (Krieg and Melton, Meth Enzymol (1987)155:397-415, which reference is hereby incorporateds by reference in itsentirety).

[0166] Oocytes from Xenopus laevis were harvested and prepared usingpublished techniques (Coleman, A., in Hames, B. D., and Higgins, S. J.,eds, Transcription and Translation: A Practical Approach, IRL Press, pp.271-302; Williams, J. A., et al. Proc Natl Acad Sci USA (1988)85:4939-4943]. To remove follicular cells, oocytes were incubated for1.5 h with shaking in calcium-free Barth's containing 2 mg/ml each ofcollagenase 1A and hyaluronidase 1S. The oocytes were then washed fivetimes in regular Barth's and incubated at 18° C. in Barth's mediumcontaining 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 mMsodium pyruvate. Stage V oocytes were selected and injected with 30 nlof cRNA (0.33 μg/μl water) or water alone, and then incubated with 0.25ml of medium in groups of four/well in a 96-well culture plate. After 36hours the oocytes were incubated with ⁴⁵Ca (250 μCi/ml). After 12 hincubation the oocytes were washed and 0.2 ml of medium added andreplaced every five minutes. The harvested medium was analyzed byscintillation counting. After five replacements to determine thebaseline release of ⁴⁵Ca, test medium with the agonist, e.g. SLIGRL, wasadded and the evoked ⁴⁵Ca-release determined.

[0167] Oocytes were injected with capped cRNA (ca 10 ng) encodingwild-type mouse C140 receptor (WT) or either of the three mutantreceptors 35Pro, 35Arg and 35His. After 36 hours, cRNA-injected andcontrol water-injected, oocytes were loaded with ⁴⁵Ca, and 12 hoursthereafter peptide or trypsin-induced ⁴⁵Ca release were determined asdescribed above. The peptide SLIGRL was added at 100 μM, and trypsin at300 pM. The stimulation with the peptide was done on the same group ofoocytes after the stimulation with trypsin. The data shown in Table 1represent the mean of three replicate determinations, and denotes theincrease compared to oocytes injected with water. TABLE 1 ReceptorAgonist Fold increase in ⁴⁵Ca WT Trypsin 6.6 35Pro Trypsin 0 35ArgTrypsin 0 35His Trypsin 0 WT SLIGRL 11 35Pro SLIGRL 23 35Arg SLIGRL 1535His SLIGRL 23

[0168] As shown in Table 1, the agonist peptide SLIGRL was able toactivate both the wild-type and mutated receptors. On the other hand,trypsin, which can activate only by cleavage of the extracellulardomain, is able only to activate the wild-type receptor.

EXAMPLE 7 Activation of the C140 Receptor by Different Agonist Peptides

[0169] Various peptides were tested at 100 μM in the assay above usingwild-type mouse C140 receptor, expressed in oocytes. The results areshown in Table 2. TABLE 2 Peptide Fold Increase in ⁴⁵Ca SLIGRL 15 SLIGRA8.5 SLIGAL 0 SLIARL 4.3 SLAGRL 0 SAIGRL 0 ALIGRL 1.3 SFFLRW 1.7

[0170] The “native” peptide SLIGRL is most effective; replacing L atposition 6 with alanine lowers but does not destroy activity. Positions2 and 3 are more sensitive. Position 1 tolerates substitution withalanine but decreases the activity by a factor of 10; the activity ofthis agonist is comparable to the analogous thrombin receptor agonistSFFLRW.

EXAMPLE 8 Expression of C140 Receptor in Various Tissues

[0171] Poly(A)+RNA was prepared from mouse tissues, resolved on a 1.2%agarose gel containing 50% formamide and blotted onto Hybond C extramembrane (Amersham). The blot was hybridized with a ³²P-labeled “randompriming probe” directed against the whole coding region of murine C140receptor. The probe was hybridized at 42° C. for 48 hr then successivelywashed at 20° C. in 1×SSC, 0.1% SDS twice, 5 min each time, then at 65°C. in 1×SSC, again twice for 20 min each time, and then 0.1×SSC, 0.1%SDS twice for 20 min each time. The resulting membrane wasautoradiographed for 5 days at −80° C. with an intensifying screen.

[0172] The results, shown in FIG. 6 indicate that kidney and smallintestine, but not spleen, contain mRNA encoding C140. In FIG. 6, whereeach lane contains 10 μg RNA, lane A is derived from spleen, lane B fromkidney and lane C from small intestine.

EXAMPLE 9 Expression of C140 Transcripts in Mice

[0173] In situ hybridization using ³⁵S RNA probes was used to localizeC140 transcripts in mouse embryogenesis and in adult mouse tissues. Astrong signal was found in the gastrointestinal tract at 11.5 days; at14 days there was strong hybridization to epithelial structures in thenasopharynx, stomach-intestine, skin and endothelial cells in largervessels. There was some hybridization in the liver and sclerotoma but nosignal in muscle or CNS. At 17 days, the signals in the sclerotoma haddisappeared and additional epithelial structures showed hybridizationincluding the esophagus, kidney glomeruli, lung, hair follicles andepidermis.

[0174] In newborns, the signals found at 17 days were retained andadditional signals were found in the thymic medulla and kidney medulla.Adults showed transcripts in the mucosa of stomach, intestine and colon,white pulp of the spleen, thymus and kidney medulla. Again, there wereno signals in the CNS, liver, lung or adrenal gland. FIG. 12 shows theresults of in situ hybridization in a sectioned newborn mouse usingthese probes.

EXAMPLE 10 Expression of C140 Transcripts in Human Tissues

[0175]FIG. 13 shows the results of a Northern blot of total RNA fromhuman cell lines hybridized to a human C140 receptor probe. Ten mg oftotal RNA was used. Hybridization was obtained in RNA from stomach (lane1), Ca—Co-2 cells (lane 2); HT-29 cells (lane 3), A498 cells (lane 5),5637 cells (lane 8); skin keratinocytes (lane 12), and HUVEC (lanes 13and 14). No hybridization was detected in HuTu80 cells, J82 cells,MCF-7, HeLa or NCI 12 cells (lanes 4, 6, 9 and 10).

EXAMPLE 11 Determination of Hypotensive Activity of C140 Agonists

[0176] The C140 agonist SLIGRL was injected in 0.2 ml buffer atvarious-concentrations into rat femoral vein and the arterial pressurewas monitored. The results of various concentrations are shown in FIG.7.

[0177] The trace in FIG. 7 shows that even at 0.1 mM an appreciabledecrease in blood pressure occurred; larger decreases were observed at 1mM concentration.

[0178] This effect was also shown by observing vasodilation as a resultof stimulation of the rat femoral vein with the above agonist. AdultSprague-Dawley rats were killed by exsanguination during diethyletheranesthesia and the femoral vein was removed and dissected free from fatand connective tissue. Circular preparations of the vein were mounted inan organ bath (5 ml) on two L-formed metal holders (0.2 mm diameter).One of the metal holders was screwed into one of the levers of a GrassFTO C force displacement transducer. The bathing liquid was Kreb'sRinger solution containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mMMgSO₄, 24.8 mM NaHCO₃, 1.2 mM KH₂PO₄ and 5.6 mM glucose. The bathingfluid was continuously treated with 88.5% oxygen-11.5% CO₂; thetemperature was held at 37° C. The endothelium was removed by bubblingCO₂ through the vessels. The basal tension was between 7.5 and 12 mN.The preparations were equilibrated for at least 1 hr before applicationof agonist and control substances.

[0179] The results of these determinations are shown in FIGS. 8a and 8b. As shown in FIG. 8a, contraction induced by application of PGF_(2α)at 3×10⁻⁵ M is relaxed by administration of 10⁻⁵ M agonist. The resultsin FIG. 8a were obtained using the vein with the endothelium stillpresent.

[0180] In FIG. 8b, the endothelium has been removed. In an analogousexperiment, the contraction induced by 3×10⁻⁵ M PGF_(2α) is notcounteracted by 10⁻⁵ M agonist or by 10⁻⁵ M acetylcholine.

EXAMPLE 8 Activation of Recombinant C140 Receptor by Plasmin andKallikrein

[0181]FIGS. 9a and 9 b show the ability of plasmin and kallikreinrespectively to activate oocytes injected with C140 cRNA (open circles)or water (crosses) as control. FIG. 9c shows the ability of trypsin toactivate frog oocytes injected with C140 receptor cRNA (filled circles)or substance K receptor cRNA (open circles). Trypsin clearly has adifferential effect on the C140 receptor-injected oocytes.

[0182] All references cited and mentioned above, including patents,journal articles and texts, are all incorporated by reference herein,whether expressly incorporated or not.

[0183] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

[0184] While this invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. A DNA molecule comprising an expression system capable, whentransformed into a recombinant host, of producing the C140 receptor atthe cell surface of the host, which expression system comprises anucleotide sequence encoding the C140 receptor operably linked to acontrol sequence heterologous to said encoding nucleotide and operablein said host cell.
 2. A cell modified to contain the expression systemof claim
 1. 3. A method to produce cells that contain C140 receptordeployed at their surface, which method comprises culturing the cells ofclaim 2 under conditions which effect the expression of the nucleotidesequence encoding the C140 receptor to obtain said cells that containC140 receptor deployed at their surface.
 4. A cRNA molecule that encodesthe C140 receptor.
 5. Cells which are oocytes modified to contain thecRNA of claim
 4. 6. A method to produce cells which are oocytes thatcontain C140 receptor deployed at their surface, which method comprisesculturing the oocytes of claim 5 under conditions which effect theexpression of the cRNA encoding the C140 receptor to obtain said cellsthat contain C140 receptor deployed at their surface.
 7. A method todetermine the C140 agonist activity of a candidate substance, whichmethod comprises: incubating the cells of claim 3 or 6 in the presenceand absence of the substance, and detecting the presence, absence oramount of activation of the C140 receptor in the presence as compared tothe absence of said substance whereby an increase in said activation inthe presence as compared to the absence of said substance indicatesagonist activity of the substance.
 8. A method to assess the ability ofa candidate substance to behave as a C140 antagonist, which methodcomprises: incubating the cells of claim 3 or 6 in the presence of aC140 agonist and in the presence and absence of said candidate, andmeasuring the activation of the C140 receptor in the presence andabsence of said candidate, whereby a decrease in said activation in thepresence of the candidate indicates the antagonist activity of thecandidate.
 9. A method to assess the ability of a candidate substance tobind to C140 receptor, which method comprises: incubating the cells ofclaim 3 or 6 in the presence of a C140 agonist or a known C140antagonist and in the presence and absence of said candidate, andmeasuring the binding of said C140 agonist or C140 antagonist to thesurface of said cells in the presence and absence of said candidate,whereby a decrease in said binding in the presence of the candidateindicates the ability of the candidate to bind receptor.
 10. An antibodycomposition specifically immunoreactive with an extracellular region ofthe C140 receptor protein or a portion thereof.
 11. The antibodycomposition of claim 10 wherein said region is the ligand-bindingregion, or which is specifically immunoreactive with activated C140receptor, or recognizes an epitope within the receptor sequence SLIGRL,or is specifically reactive with the cleaved activation peptide of theC140 receptor.
 12. A method to localize activated C140 receptors insitu, which method comprises: administering to a subject putativelyharboring activated C140 receptor an amount of antibody specific to saidactivated receptor effective to bind to said activated receptor, anddetecting the location of said antibody.
 13. A method for detecting thepresence of activated C140 receptor in a mammalian subject, which methodcomprises: contacting a sample of the biological fluid of said subjectwith a detection system which measures the presence, absence or amountof the cleaved activation peptide of the C140 receptor; and detectingthe presence, absence or amount of said cleaved peptide.
 14. An agonistpeptide capable of activating C140 receptor, which peptide is of theformula AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-Z   (1) wherein AA₁ is a small aminoacid or threonine; AA₂ and AA₃ are each independentlyneutral/nonpolar/large/nonaromatic amino acids; AA₄ is a small aminoacid; AA₅ is a basic amino acid; AA₆ may be present or absent and, ifpresent, is a neutral/nonpolar/large/nonaromatic amino acid; AA₇ isabsent if AA₆ is absent and may be present or absent if AA₆ is present,and is an acidic amino acid; and Z is a substituent that does notinterfere with agonist activity.
 15. The peptide of claim 14 wherein AA₁is ser, ala, gly, thr, or 2,3-diamino-propionic (2,3-diaP); and/orwherein each of AA₂ and AA₃ is independently selected from the groupconsisting of ile, val, leu, and Cha; and/or wherein AA₄ is Gly; and/orwherein AA₅ is Arg, Lys or Har; and/or wherein Z comprises OR′, or NR′R′wherein each R′ is independently H or is a straight or branched chainalkyl or 1-6C, wherein each R′ may optionally be substituted with one ormore substituents selected from the group consisting of —OR′, —NR′R′,and —NR′CNR′NR′R′ wherein each R′ is H or is a straight or branchedchain alkyl of 1-6C.
 16. The peptide of claim 15 wherein AA₁-AA₂-AA₃ isselected from the group consisting of SLI, SLL, SChaI, SChaL,(2,3-diaP)LI and (2,3-diaP)LL; and/or wherein Z includes additionalpeptide sequence of 1-5 amino acids.
 17. The peptide of claim 14 whichis selected from the group consisting of SLIGRLETQPPIT, SLIGRLETQPPI,SLIGRLETQPP, SLIGRLETQP, SLIGRLETQ, SLIGRLET, SLIGRLE, SLIGRL, SLIGR,SLLGKVDGTSHVT, SLLGKVDGTSHV, SLLGKVDGTSH, SLLGKVDGTS, SLLGKVDGT,SLLGKVDG, SLLGKVD, SLLGKV, SLLGK, S(Cha)IGR, S(Cha)LGK, (2,3-diaP)-LIGR,(2,3-diaP)LLGK, SLLGKR—NH₂, SLIGRR—NH₂, S(Cha)LGKK—NH₂, S(Cha)IGRK—NH₂,(2,3-diaP)-LIGRK—NH₂, and (2,3-diaP)-LLGKK—NH₂.
 18. A peptide capable ofinhibiting the function of the C140 receptor which peptide is of theformula X-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-Z wherein X is an amino acid residueother than ser, ala, thr, cys, 2,3-diaP or gly or is a desamino oracylated amino acid, wherein AA₂ and AA₃ are each independentlyneutral/nonpolar/large/nonaromatic amino acids; AA₄ is a small aminoacid; AA₅ is a basic amino acid; AA₆ may be present or absent and, ifpresent, is a neutral/nonpolar/large/nonaromatic amino acid; AA₇ isabsent if AA₆ is absent and may be present or absent if AA₆ is present,and is an acidic amino acid; and Z is a substituent that does notinterfere with agonist activity.
 19. The peptide of claim 18 wherein Xis Mvl, Mpr, Mba, or SMeMpr; and/or wherein each of AA₂ and AA₃ isindependently selected from the group consisting of ile, val, leu, Nle,Nva, Cyclopentylalanine and Cha; and/or wherein AA₄ is Gly; and/orwherein AA₅ is Arg, Lys, Orn or Har; and/or wherein Z comprises OH or anester or salt thereof, or NR′R′ wherein each R′ is independently H or isa straight or branched chain alkyl of 1-6C, wherein each R′ mayoptionally be substituted with one or more substituents selected fromthe group consisting of —OR′, —NR′R′, and —NR′CNR′NR′R′ wherein each R′is H or is a straight or branched chain alkyl of 1-6C.
 20. The peptideof claim 19 wherein AA₂-AA₃ is selected from the group consisting of LI,LL, ChaI, and ChaL; and/or wherein Z includes a peptide extension of 1-5amino acid residues.
 21. The peptide of claim 18 which is selected fromthe group consisting of Mpr-LLGK, Mpr-LIGR, Mpr-(Cha)LKG, Mpr-(Cha)IGR,Mpr-LLGKK-NH₂, Mpr-LIGRK-NH₂, Mpr-LIGRKETQP-NH₂, Mpr-LLGKKDGTS-NH₂,(n-pentyl)₂-N-Leu-Ile-Gly-Arg-Lys-NH₂ and(Me-N-(n-pentyl)-Leu-Ile-Gly-Arg-Lys-NH2, and the amidated or acylatedforms thereof.
 22. An isolated nucleic acid molecule which encodes aC140 receptor polypeptide or which is complementary to a DNA or RNAmolecule encoding a C140 receptor polypeptide.
 23. The nucleic acidmolecule of claim 22 wherein said C140 receptor is the human C140receptor.
 24. A method to inhibit expression of C140 receptors in a cellcomprising providing to said cell an oligonucleotide molecule which isantisense to, or forms a triple helix with, C140 receptor-encoding DNAor with DNA regulating expression of C140 receptor-encoding DNA, in anamount sufficient to inhibit expression of said C140 receptors, therebyinhibiting said expression.
 25. A method to inhibit expression of C140receptors in a subject, comprising administering to said subject anoligonucleotide molecule which is antisense to, or forms a triple helixwith, C140 receptor-encoding DNA or with DNA regulating expression ofC140 receptor-encoding DNA, in an amount sufficient to inhibitexpression of said C140 receptors in said subject, thereby inhibitingsaid expression.
 26. A pharmaceutical composition comprising anoligonucleotide molecule of claim 25 together with a pharmaceuticallyacceptable carrier or excipient.